Altered survival signaling is a hallmark of malignant melanoma cells resulting in resistance to drug-induced apoptosis. Previous studies suggest that cancer cell survival depends on the modulation of cell death pathways by methylglyoxal (MG), a reactive by-product of glycolysis that acts by posttranslational modification and functional alteration of target proteins involved in cellular survival. Our preliminary data indicate that heat shock protein 27 (Hsp27) is the exclusive target of MG-adduction in human melanoma and that MG-antagonists selectively induce apoptosis and chemosensitization in human melanoma cells. In this proposal we will test the hypothesis that the glycolytic control of melanoma cell survival is mediated by MG adducted heat shock protein 27 (MG-Hsp27), a novel therapeutic target amenable to small molecule modulation by specific MG-antagonists. First, MG-Hsp27 will be characterized at the molecular, cellular, and tissue level by mapping the human melanoma MG-Hsp27 proteome (specific aim #1). The exact structure and site(s) of human melanoma Hsp27 posttranslational modification by phosphorylation and MG-adduction will be determined in melanoma cell lines and tissue. MG-Hsp27 will then be validated as a molecular target for the modulation of melanoma cell survival (specific aim #2). The molecular mechanism of survival signaling by MG-Hsp27 will be elucidated, and target validation will be achieved by metabolic, pharmacological, and genetic modulation of MG-Hsp27. Next, cell-based screening of small molecule MG antagonists for potency of target modulation and anti-melanoma activity will identify a lead series of bioactive MG-Hsp27-inhibitors (specific aim #3). Finally, proof-of-principle evidence for chemotherapeutic potential of MG-antagonists will be tested in a xenograft mouse melanoma model using MG-antagonists as single agents or in combination with other chemotherapeutic agents (specific aim #4). Successful completion of the proposed research will identify a unique metabolic vulnerability of cancer cells that can be attacked by small molecule antagonists. Lay description: Melanoma, a highly aggressive tumor that originates from pigment producing cells in human skin, has an increasing incidence that currently surpasses that of any other cancer. My recent research suggests that a byproduct of sugar energy metabolism called methylglyoxal may represent an Achilles heel of human melanoma cells. The proposed research aims to elucidate the molecular mechanism that regulates melanoma cell survival by methylglyoxal and to test antagonists of methylglyoxal as novel anti-melanoma therapeutics.